The present invention relates to a method of providing enzyme immobilized products which can be continuously and repeatedly used in reactions employing enzymes.
In recent years, the industrial use of specific catalytic reactions of enzymes as sensors and chemical production is performed with the aid of developments in the enzyme immobilizing art. As conventional methods for immobilization of enzymes, there are known a entrapping method of performing immobilization by entrapment within interstitial space of macromolecular matrices, a support bonding method for directly making chemical bond with the immobilizing support, a crosslinking method for crosslinking enzymes with respect to each other, or the like.
Concretely, various organic macromolecules such as resin, sepharose, etc. or inorganic substances such as glass beads, metal, carbon are used as a support for the immobilization of enzymes. The enzyme is immobilized on these supports, using the above-described immobilizing methods. According to the support bonding method, the immobilizing process is complicated and applicable supports are limited. Also, according to the entrapping method, the immobilizing operation is performed by using photo-polymerization or the like to confine the enzyme in the matrices of resin. According to this method, the enzyme activity decline caused by immobilization is small, since the enzyme can be immobilized on the support without chemical modification is inevitably lost through the continuous and repeated use.
According to the crosslinking method, an immobilizing reagent such as glutaraldehyde or the like is used to crosslink the enzymes with respect to each other to immobilize them. Concretely, the surface of the immobilizing support such as glass plate, glass beads, etc. is covered with enzyme solution by spreading, immersing and is dried if necessary. Then, an immobilizing reagent solution such as glutaraldehyde or the like is added to cause the crosslinking reaction thereby to immobilize the enzyme. A several percent dilute solution of glutaraldehyde is generally used. The crosslinking reaction abruptly advances with high immobilizing-reagent concentration whereby the decrease of the enzyme activity is caused. The reaction rate is slow with low immobilizing reagent concentration. During this period, unreacted enzyme is dissolved in the added immobilizing-reagent solution and is lost from the support. This is extremely disadvantageous for promoting the reaction. Therefore, it is almost impossible to uniformly cover the support surface with the membrane of the immobilized enzyme by this method. Since this is a method of applying the immobilizing reagent in as a solution, namely, supplying the immobilizing reagent from a liquid phase when the immobilizing reaction (crosslinking reaction) is performed, the above-described problems are unavoidable.